鸡传染性喉气管炎病毒gB基因的克隆及其在耻垢分枝杆菌中的表达

以ILTV基因组为模板 ,利用PCR特异扩增出gB基因 ,定向克隆到中间质粒载体pY_α ,构建了中间质粒pY_α_gB。然后以中间质粒pY_α_gB为模板 ,扩增出含有人结核分枝杆菌启动子hsp70基因和堪萨斯分枝杆菌α信号肽基因的hsp_α_gB片段 ,回收补平后与穿梭表达载体pRR3平端连接 ,从而构建大肠杆菌_分枝杆菌穿梭表达质粒pR_α_gB。再将其电转化至耻垢分枝杆菌M .smegmatismc2 15 5 ,ELISA检测表明重组菌株M .smegmatismc2 15 5 (pR_α_gB)的表达产物具有很好的反应原性。Westernblot检测说明gB基因在分枝杆菌中获得了表达并具有良好的免疫原性。鸡胚中和试验结果表明该重组菌株可以中和 1个剂量EID50 的ILTV强毒 ,能够保护SPF鸡胚抵抗强毒攻击     

大鼠脂肪间充质干细胞诱导为类肝细胞及其细胞片的制备

成体多能干细胞,如来自骨髓和脂肪组织的间充质干细胞等具有多向分化的潜能。虽然自体干细胞移植已经发展成为器官移植的有效代替疗法之一,但是由于移植位点细胞的流失和分化条件的限制等问题使得这种疗法的效率大大降低。本研究目的是将由脂肪干细胞分化而来的类肝细胞制备成具有稳定细胞性状的可移植的肝细胞片。首先在体外分离扩增脂肪干细胞,并通过控制严格地分化条件获得类肝细胞。然后将此细胞接种到聚N-异丙基丙烯酰胺(PNIPAAm)结合的细胞培养皿表面,通过调节培养温度到20oC,使细胞成片脱离培养皿形成细胞片。对细胞片进行了常规HE染色和免疫组化观察,结果显示:这类细胞片中平均含有2～3层细胞,并且保持了细胞外基质的完整。同传统的胰酶消化收集移植用细胞相比,细胞片方法极大地减少了对移植用细胞的细胞膜和细胞外基质的损伤,这将大大促进细胞片和原位组织的相互作用,增加细胞利用效率,从而有望提高治疗效果。     

Wangella harbinensis gen. nov., sp. nov., a new member of the family Micromonosporaceae

A novel endophytic actinomycete, designated strain NEAU-J3^T, was isolated from soybean root (Glycine max (L.) Merr) and characterized using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences suggested that strain NEAU-J3^T fell within the family Micromonosporaceae. The strain was observed to form an extensively branched substrate mycelium, which carried non-motile oval spores with a smooth surface. The cell walls of strain NEAU-J3^T were determined to contain meso-diaminopimelic acid and galactose, ribose and glucose were detected as whole-cell sugars. The major menaquinones were determined to be MK-9(H₄) and MK-9(H₆). The phospholipids detected were phosphatidylcholine and phosphatidylethanolamine. The major cellular fatty acids were determined to be C_16:0, C_18:1 ω9c, C_18:0, C_17:0, C_17:1 ω7c, anteiso-C_17:0, C_16:1 ω7c and C_15:0. The DNA G + C content was 62.5 mol%. On the basis of the morphological and chemotaxonomic characteristics, phylogenetic analysis and characteristic patterns of 16S rRNA gene signature nucleotides, strain NEAU-J3^T is considered to represent a novel species of a new genus within the family Micromonosporaceae, for which the name Wangella harbinensis gen. nov., sp. nov. is proposed. The type strain of Wangella harbinensis is strain NEAU-J3^T (=CGMCC 4.7039^T = DSM 45747^T).     

Pyrosequencing Reveals Contrasting Soil Bacterial Diversity and Community Structure of Two Main Winter Wheat Cropping Systems in China

Microbes are key components of the soil environment, playing an important role in maintaining soil health, sustainability, and productivity. The composition and structure of soil bacterial communities were examined in winter wheat–rice (WR) and winter wheat–maize (WM) cropping systems derived from five locations in the Low-Middle Yangtze River plain and the Huang-Huai-Hai plain by pyrosequencing of the 16S ribosomal RNA gene amplicons. A total of 102,367 high quality sequences were used for multivariate statistical analysis and to test for correlation between community structure and environmental variables such as crop rotations, soil properties, and locations. The most abundant phyla across all soil samples were Proteobacteria, Acidobacteria, and Bacteroidetes. Similar patterns of bacterial diversity and community structure were observed within the same cropping systems, and a higher relative abundance of anaerobic bacteria was found in WR compared to WM cropping systems. Variance partitioning analysis revealed complex relationships between bacterial community and environmental variables. The effect of crop rotations was low but significant, and interactions among soil properties, locations, and crop rotations accounted for most of the explained variation in the structure of bacterial communities. Soil properties such as pH, available P, and available K showed higher correlations (positive or negative) with the majority of the abundant taxa. Bacterial diversity (the Shannon index) and richness (Chao1 and ACE) were higher under WR than WM cropping systems.     

Inhibition of Filamin-A Reduces Cancer Metastatic Potential

Filamin-A cross-links actin filaments into dynamic orthogonal networks, and interacts with an array of proteins of diverse cellular functions. Because several filamin-A interaction partners are implicated in signaling of cell mobility regulation, we tested the hypothesis that filamin-A plays a role in cancer metastasis. Using four pairs of filamin-A proficient and deficient isogenic cell lines, we found that filamin-A deficiency in cancer cells significantly reduces their migration and invasion. Using a xenograft tumor model with subcutaneous and intracardiac injections of tumor cells, we found that the filamin-A deficiency causes significant reduction of lung, splenic and systemic metastasis in nude mice. We evaluated the expression of filamin-A in breast cancer tissues by immunohistochemical staining, and found that low levels of filamin-A expression in cancer cells of the tumor tissues are associated with a better distant metastasis-free survival than those with normal levels of filamin-A. These data not only validate filamin-A as a prognostic marker for cancer metastasis, but also suggest that inhibition of filamin-A in cancer cells may reduce metastasis and that filamin-A can be used as a therapeutic target for filamin-A positive cancer.     

Combinatorial signals of activin/nodal and bone morphogenic protein regulate the early lineage segregation of human embryonic stem cells

Cell fate commitment of pre-implantation blastocysts, to either the inner cell mass or trophoblast, is the first step in cell lineage segregation of the developing human embryo. However, the intercellular signals that control fate determination of these cells remain obscure. Human embryonic stem cells (hESCs) provide a unique model for studying human early embryonic development. We have previously shown that Activin/Nodal signaling contributes to maintaining pluripotency of hESCs, which are derivatives of the inner cell mass. Here we further demonstrate that the inhibition of Activin/Nodal signaling results in the loss of hESC pluripotency and trophoblast differentiation, similar to BMP4-induced trophoblast differentiation from hESCs. We also show that the trophoblast induction effect of BMP4 correlates with and depends on the inhibition of Activin/Nodal signaling. However, the activation of BMP signaling is still required for trophoblast differentiation when Activin/Nodal signaling is inhibited. These data reveal that the early lineage segregation of hESCs is determined by the combinatorial signals of Activin/Nodal and BMP.     

Packing of the transmembrane helices of Na,K-ATPase: direct contact between beta-subunit and H8 segment of alpha-subunit revealed by oxidative cross-linking

Spatial relationships among the transmembrane (TM) segments of alpha- and beta-subunits of the Na,K-ATPase molecule have been investigated using oxidative induction of disulfide bonds. The catalytic alpha-subunit contains 10 TM alpha-helices (H1-H10) with 9 Cys residues located within or close to the membrane moiety. There is one Cys residue in the single TM segment of beta-subunit (Hbeta). Previously, the cross-linking products containing the beta-subunit and two fragments of alpha-subunit (the N-terminal containing H1-H2 helices and the C-terminal containing H7-H10 helices) have been identified in experiments with membrane-bound or detergent-solubilized preparations of the membrane moiety of trypsin-digested Na,K-ATPase [Sarvazyan, N. A., Modyanov, N. N., and Askari, A. (1995) J. Biol. Chem. 270, 26528-26532 and Sarvazyan, N. A., Ivanov, A., Modyanov, N. N., and Askari, A. (1997) J. Biol. Chem. 272, 7855-7858]. Here, we have shown that Cu(2+)-phenanthroline treatment of digitonin-solubilized preparation provides the most efficient formation of intersubunit cross-linked product that is predominantly a dimer of beta-subunit and a 22-kDa C-terminal alpha-fragment containing H7-H10 helices. This cross-linked product was isolated and subjected to CNBr cleavage. The resulting fragments were electrophoretically separated and sequenced. A 17-kDa peptide composed of Ile853-Met942 alpha-fragment and Ala5-Met56 beta-fragment was identified as a product of intersubunit disulfide cross-link between Cys44 of Hbeta and either Cys911 or Cys930, located in H8. This provides the first direct experimental evidence of the juxtaposition of Hbeta and H8 within the Na,K-ATPase molecule. The second detected cross-linked product was composed of alpha-fragments Lys947-Met963 and Tyr974-Tyr1016 linked by induced disulfide bridge between Cys964 (H9) and Cys983 (H10). The spatial proximity of these Cys residues defines the mutual orientation of H9 and H10 helices of alpha-subunit.     

蜡梅种子抑菌成分研究

在抑菌活性追踪指导下,采用硅胶柱层析的方法。从蜡梅种子中分离得到一活性化合物A,经质谱、核磁共振波谱等技术鉴定为d－洋蜡梅碱。经杀虫活性和抑菌活性测定,该化合物对粘虫的幼虫无毒杀活性;对西瓜枯萎病菌、玉米小斑病菌、玉米大斑病菌、番茄早疫病菌均有显著的抑菌活性。其抑制中浓（EC50）分别为：3878．8、29．3、103．1、328．3mg／L,但对油菜菌核病菌无抑制活性。